Modern silviculture often requires the planting of large numbers of genetically identical plants that have been selected to have advantageous properties. Production of new plants by sexual reproduction, which yields botanic seeds, is usually not feasible. Asexual propagation, via the culturing of somatic or zygotic embryos, has been shown for some species to yield large numbers of genetically identical embryos, each having the capacity to develop into a normal plant.
Somatic cloning is the process of creating genetically identical plants from plant tissue other than male and female gametes. In one approach to somatic cloning, plant tissue is cultured in an initiation medium that includes hormones, such as auxins and/or cytokinins, to initiate formation of embryogenic tissue, such as embryogenic suspensor masses, that are capable of developing into somatic embryos. The embryogenic tissue is then further cultured in a multiplication medium that promotes establishment and multiplication of the embryogenic tissue to form pre-cotyledonary embryos (i.e., embryos that do not possess cotyledons). The pre-cotyledonary embryos are then cultured in a development medium that promotes development and maturation of cotyledonary somatic embryos that can, for example, be placed on germination medium to produce germinants, and subsequently transferred to soil for further growth, or alternatively, placed within manufactured seeds and sown in soil where they germinate to yield seedlings. Manufactured seeds are described, for example, in U.S. Pat. Nos. 5,564,224; 5,687,504; 5,701,699; and 6,119,395.
The somatic embryogenesis process typically is laborious and inefficient. For example, one of the steps in the process involves movement of embryogenic tissue from liquid multiplication media and subsequent plating at low density on a semi-solid media surface for embryo development and maturation. This step is typically done manually by a skilled technician using a pipette to dispense a mixture of embryogenic cells and liquid medium onto development medium.
Another labor intensive step in the embryogenesis process is the selective harvesting from development medium of individual embryos suitable for germination. At the end of the development phase, the embryos may be present in a number of stages of maturity and development. Those that are most likely to successfully germinate into normal plants are preferentially selected using a number of visually evaluated screening criteria such as the embryo's size, shape (e.g., axial symmetry), cotyledon development, surface texture, color, and others, and manually plucked out of the development medium with a pair of forceps. The selected desirable embryos are then carefully laid out, and separated from each other for further processing. This is a highly skilled yet tedious job that is time consuming and expensive. Further, it poses a major production bottleneck when the ultimate desired output is in the millions of plants.
Efforts have been made to automate the somatic embryogenesis process. Scale-up and automating somatic embryogenesis technology may involve the use of large volumes of liquid media or water for purposes of dilution and/or singulation of immature and mature embryos in order to move and position the embryos for subsequent process steps. For example, suspension cultures at the end of the multiplication stage may be diluted in order to facilitate even plating of the pre-cotyledonary embryos onto development medium.
Another example of the use of large volumes of liquid is in the singulation step. Singulation is a processing step that occurs at the end of development and maturation in which embryos are physically separated from each other and the underlying embryogenic suspensor mass (ESM) before further processing such as, for example, insertion into manufactured seed, or placement onto germination or pre-germination medium for further treatment prior to insertion into manufactured seed. Singulation may be accomplished by spraying the embryos and attached ESM with liquid to remove them from the development medium; using a series of sieves to separate the embryos from each other and residual ESM; placing the embryos into large volumes of liquid; and subsequently placing individual embryos onto a porous substrate.
The presence of excess liquid on the substrate on which the embryos are disposed at the plating step and/or singulation step can be problematic. Avoiding excess wetness and retention of liquid medium hormone residues at the gel-cell interface is critical for quality embryo development. Furthermore, the presence of liquid on the substrate on which the embryos are disposed can have significant negative effects on germination.
Therefore methods are needed to remove liquid from the surface of embryos and the substrate on which embryos are disposed, without harming the embryos or disturbing the position of the embryos on the substrate. The present invention addresses these and other needs.